Yeast Rim11 kinase responds to glutathione-induced stress by regulating the transcription of phospholipid biosynthetic genes.

Glutathione (GSH), a tripeptide composed of glycine, cysteine, and glutamic acid, is an abundant thiol found in a wide variety of cells, ranging from bacterial to mammalian cells. Adequate levels of GSH are essential for maintaining iron homeostasis. The ratio of oxidized/reduced GSH is strictly regulated in each organelle to maintain the cellular redox potential. Cellular redox imbalances cause defects in physiological activities, which can lead to various diseases. Although there are many reports regarding the cellular response to GSH depletion, studies on stress response to high levels of GSH are limited. Here, we performed genome-scale screening in the yeast Saccharomyces cerevisiae and identified RIM11, BMH1, and WHI2 as multicopy suppressors of the growth defect caused by GSH stress. The deletion strains of each gene were sensitive to GSH. We found that Rim11, a kinase important in the regulation of meiosis, was activated via autophosphorylation upon GSH stress in a glucose-rich medium. Furthermore, RNA-seq revealed that transcription of phospholipid biosynthetic genes was downregulated under GSH stress, and introduction of multiple copies of RIM11 counteracted this effect. These results demonstrate that S. cerevisiae copes with GSH stress via multiple stress-responsive pathways, including a part of the adaptive pathway to glucose limitation.

TAQing2.0 for genome reorganization of asexual industrial yeasts by direct protein transfection.

Genomic rearrangements often generate phenotypic diversification. We previously reported the TAQing system where genomic rearrangements are induced via conditional activation of a restriction endonuclease in yeast and plant cells to produce mutants with marked phenotypic changes. Here we developed the TAQing2.0 system based on the direct delivery of endonucleases into the cell nucleus by cell-penetrating peptides. Using the optimized procedure, we introduce a heat-reactivatable endonuclease TaqI into an asexual industrial yeast (torula yeast), followed by a transient heat activation of TaqI. TAQing2.0 leads to generation of mutants with altered flocculation and morphological phenotypes, which exhibit changes in chromosomal size. Genome resequencing suggested that torula yeast is triploid with six chromosomes and the mutants have multiple rearrangements including translocations having the TaqI recognition sequence at the break points. Thus, TAQing2.0 is expected as a useful method to obtain various mutants with altered phenotypes without introducing foreign DNA into asexual industrial microorganisms.

Micrococcus luteus K-3-type glutaminase from Aspergillus oryzae RIB40 is salt-tolerant.

Aspergillus oryzae RIB40 possesses the gene of glutaminase (Micrococcus luteus K-3-type glutaminase; AoGls), which has 40% homology with the salt-tolerant glutaminase from M. luteus K-3 (Micrococcus glutaminase). It was found that AoGls is a salt-tolerant enzyme, and its properties are similar to those of Micrococcus glutaminase.

Molecular cloning, overexpression, and purification of Micrococcus luteus K-3-type glutaminase from Aspergillus oryzae RIB40.

We have for the first time found and cloned the cDNA (AoglsA) of Aspergillus oryzae RIB40, which encodes a 49.9-kDa protein sharing 40% homology with the salt-tolerant glutaminase of Micrococcus luteus K-3 (Micrococcus glutaminase). AoglsA was subcloned into a series of expression vectors and expressed in Saccharomyces cerevisiae and Escherichia coli. The gene product, which we named AoGls, showed glutaminase activity and was produced in a cell wall fraction of S. cerevisiae and a soluble protein in E. coli. The highest expression level of 186 U/mg was obtained when the AoglsA was inserted into six bases downstream of the Shine-Dalgarno (SD) sequence of pKK223-3 and expressed in E. coli Rosetta (DE3). AoGls was purified by SuperQ-TOYOPEARL, glutamine affinity chromatography, and Butyl-TOYOPEARL. This is the first report on the overexpression and purification of a M. luteus K-3-type glutaminase cloned from an eucaryote.

Digestion by serine proteases enhances salt tolerance of glutaminase in the marine bacterium Micrococcus luteus K-3.

Salt-tolerant glutaminase (Micrococcus glutaminase, with an apparent molecular mass of 48.3 kDa, intact glutaminase) from the marine bacterium Micrococcus luteus K-3 was digested using protease derived from M. luteus K-3. The digestion products were a large fragment (apparent molecular mass of 38.5 kDa, the glutaminase fragment) and small fragments (apparent molecular mass of 8 kDa). The digestion was inhibited by phenylmethanesulfonyl fluoride (PMSF). Digestion of intact glutaminase by serine proteases including trypsin, elastase, lysyl endopeptidase, and arginylendopeptidase also produced the glutaminase fragment. The N-terminus of the glutaminase fragment was the same as that of intact glutaminase. The N-termini of two small fragments were Ala394 and Ala396, respectively. The enzymological and kinetic properties of the glutaminase fragment were almost the same as those of intact glutaminase except for salt-tolerant behavior. The glutaminase fragment was a higher salt-tolerant enzyme than the intact glutaminase, suggesting that Micrococcus glutaminase is digested in the C-terminal region by serine protease from M. luteus K-3 to confer salt tolerance on glutaminase.